In analytical chemistry, liquid and gas chromatography techniques have become important tools in the identification of chemical sample components. The basic principle underlying all chromatographic techniques is the separation of a sample chemical mixture into individual components by transporting the mixture in a moving fluid through a porous retentive media. The moving fluid is referred to as the mobile phase and the retentive media has been referred to as the stationary phase. One of the differences between liquid and gas chromatography is that the mobile phase is either a liquid or a gas, respectively.
Consider, for example, a gas chromatograph. Typically, a supply of inert carrier gas (mobile phase) is continually passed as a stream through a heated column containing porous sorptive media (stationary phase). A sample of the subject mixture is injected into the mobile phase stream and passed through the column. A detector, positioned at the outlet end of the column, detects each of the separated components as they exit the column. Separation is due primarily to differences in the volatility characteristics of each sample component at the temperature in the column.
The analytical choice between liquid and gas chromatography techniques is largely dependent on the molecular weight of the compound being analyzed. Liquid chromatographs are capable of analyzing much heavier compounds than gas chromatographs. However, since gas chromatography detection techniques are more sensitive, they are preferred.
The advent of Supercritical Fluid Chromatography (SFC) provided a potential bridge between gas and liquid chromatography advantages, i.e., high sensitivity and heavier molecular weight samples. In SFC, a fluid heated above the critical point, is used as the mobile phase. Such fluid is passed under pressure through a media which differentially retains sample components. As the pressure of the mobile phase is increased, for example, from about 40 ATM to approximately 400 ATM, the sample being analyzed separates into its various components dependent upon the relative differential solubility of each component with the mobile phase. Since the mobile phase is a gas, gas chromatography detectors can be utilized, significantly enhancing detection sensitivity and selectivity. SFC can be simplistically viewed as an extension of gas chromatography to higher molecular weight components. SFC has been found to be primarily useful in the analysis of moderate molecular weight homologous series (M.W. 100 to 10,000) and some thermally labile molecules such as pesticides and pharmaceuticals.
Due to problems with analysis times and resolution, SFC as an analytical tool has not achieved its full potential. The term "resolution" as used herein refers to the distinctiveness with which component peaks appear in an SFC chromatogram. Chromatograms are those graphs of detector output signal vs. time which are produced by the connection of an X-Y plotter or other chart recorder to a chromatograph detector, wherein a peak indicates the detection of a component.
Originally, SFC systems used media packed columns similar to those used in liquid chromatography. However, due to concerns over large pressure drops, decreased efficiency and poor media stability, the use of capillaries in place of media packed columns as the stationary phase has become generally accepted. SFC systems utilizing a stationary phase coated capillaries are generally referred to as capillary SFC systems. Systems of this type will find the present invention of primary usefulness.
Consider generally the block diagram of the capillary SFC system shown in FIG. 1. A mobile phase fluid is supplied to a pump which in turn supplies the fluid to an injector. The sample to be analyzed is injected into the mobile phase and the combination is supplied to a splitter. Since capillary SFC utilizes a very small sample volume, typically on the order of 10 Nanoliters, a splitter is incorporated to ensure that only the allowed volume of the combination is passed through the capillary for analysis. Since SFC is carried out under predetermined pressure conditions, either static or programmed dynamic pressure conditions, a restrictor is placed at the capillary outlet and at the non-analyzed splitter output to limit mass flow. Pressure in this system is controlled by the pump. The capillary restrictor output is passed through a detector, which as mentioned previously, is preferably a gas chromatography type detector, such as a Flame Ionization Detector (FID). The output of the detector, a voltage signal, is applied to an X-Y plotter or some form of chart recorder which graphs detector output vs. time. Since pressure is also changed over time, for example, increasing the pump pressure at a fixed rate, the SFC chromatogram is also a reflection of detector output vs. pressure.
As will be understood, the splitter design and the restrictors function to create a flow ratio such that the nanoliter capillary volume is maintained. This ratio is also referred to as the split ratio.
As mentioned previously, SFC has not reached its maximum potential because it exhibits substantially lower speed and less resolution than gas chromatography. It can be shown that at the same theoretical efficiency, (i.e. numbers of theoretical plates), on the same diameter column, SFC systems are approximately 1000 times slower than gas chromatographs. By using even a smaller internal diameter capillary, SFC can be shown to be approximately 200 times slower than gas chromatography on larger capillary columns. Attempts to resolve the time problem with SFC systems typically have involved operation of smaller diameter columns at many times optimum linear velocity and modifying pressure programming, i.e., increasing the pressure at different rates during the test cycle. Unfortunately, these approaches significantly reduce resolution in known SFC systems. FIG. 2 is an example of such resolution reduction. The peaks which should be distinctly detected (dashed lines) in the latter stages of the analysis are lost in an aggregate signal.
The primary reason for such poor resolution is the effect which programmed pressure schemes and system design have on the linear velocity of the mobile phase solution. It was previously thought that capillary linear velocity remained constant during pressure programmed tests. However, it has been shown that this is not true. What may not be generally acknowledged is that as linear velocity increases, SFC efficiency decreases significantly. The most widely used capillary columns in SFC have an internal diameter of 50 um. Typical solute diffusion coefficients center around 1.times.10.sup.-4 sq.cm/sec. Using the Golay equation, uncorrected for gas non-idealities, a simple graph of theoretical plates vs. column linear velocity can be generated as in FIG. 3, showing the significant decrease in column efficiency with increasing linear velocity.
More particularly, as pump pressure pump is increased, the pressure drop across the restrictor increases. Increasing the pressure drop across the restrictor causes mass flow to increase. If the density and viscosity of the mobile phase were constant at all pressures, mass flow would increase in direct proportion to pressure. However, density and viscosity and the ratio of the two are not constant with pressure but change greatly. The combination of pressure and the ratio of density to viscosity mean that mass flow can increase enormously over the pressure range typically encountered in a pressure programmed run.
Increasing pressure increases column density at the same time. This has the effect of decreasing column linear velocity at a fixed mass flow rate, mitigating some of the effects of greatly increased mass flow through the restrictor. However, programming from 80 to 400 ATM can increase mass flow rate more than 50 times and column linear velocity more than 10 times. Such increases will not only greatly reduce the time a component remains in the capillary [retention time] and chromatogram peak widths, but will also degrade resolution.
Consequently, there is a significant need in capillary SFC to control capillary linear velocity and pressure independently, particularly during pressure programmed operation. Present instrumentation controls pressure or density only. Independent adjustment of linear velocity would allow a constant column efficiency and resolution and could also allow adjustment of a speed/resolution trade-off in different parts of the chromatogram.
Since solute diffusion coefficients in SFC are on the order of 1.times.10.sup.-4 sq.cm./sec, the optimum linear velocity of mobile phase through a capillary column can be shown to be in the range of 0.1 to 0.5 cm/sec. Higher linear velocity decreases chromatographic resolution, degrading performance. For modest resolution chromatography on a 10 meter.times.50 um ID column, maximum linear velocity should be no more than 10 cm/sec. Between these limits the mass flow rate of the mobile phase is between 0.5 and 1000.times.10.sup.-6 g/sec. As is known, such optimum linear velocities can be determined utilizing Van Deemter plots.
Since mobile phase density increases with pressure, mass flow must increase in order to maintain constant column (capillary) linear velocity since: EQU u=F/(.sub..rho. A)
where u=average linear velocity in the column in cm/sec; F=mass flow rate in g/sec; .rho.=density of the mobile phase in the column in g/cubic cm; and A=column cross sectional area in sq. cm. An ideal mass flow controller in an SFC system similar to that shown in FIG. 1 would allow adjustment of mass flow to increase, maintain or even decrease linear velocity regardless of head (pump) pressure.
Existing adjustable mechanical flow controllers tend to have large dead volumes. Since capillary SFC columns can produce solute peak volumes (volume of mobil phase containing 99% of a solute at the column exit) of less than 100 nanoliters, any flow controller must have a dead volume less than 20 nanoliters to avoid degrading the separation. In addition, flow controllers for capillary SFC must typically be mounted inside the base of gas chromatographic detectors where temperatures can be quite high. Typical temperatures of 200.degree. to 400.degree. C. and even 800.degree. C. are employed. A flow controller must be capable of operating over this temperature region. There are no viable adjustable flow controllers available with the desired characteristics.
In the absence of an adjustable flow controller, fixed geometry restrictors such as pinholes or 5-20 cm long.times.4-10 um, ID tubes are widely used. Capillary tubes drawn down to pinholes with short-to-long internal tapers are also widely used.
However, different restrictors have different mass flow characteristics dependent upon operating conditions. Consequently, if one wishes to operate under similar efficiencies for different operating conditions, restrictors should be changed. If restrictors are not changed as column pressure is increased during a test run, initially there will be a slight decrease followed by a rapid increase in linear velocity. Since it is not possible to change restrictors with different characteristics (e.g., different length and internal diameter), as pressure is increased, compromises have been made heretofore with regard to resolution and time.